Thiamine, also known as vitamin B 1, is a member of the water-soluble B-complex of vitamins and is a nutritional requirement for mammals. In nature, thiamine exists in multiple phosphorylation states: thiamine, thiamine monophosphate (TMP) and thiamine diphosphate (TPP) (there are also traces of thiamine triphosphate found in cells). All living organisms use thiamine, but thiamine and its active form thiamine pyrophosphate (TPP) are only synthesized in bacteria, fungi, and plants. Animals depend on their diet for a source of thiamin, and thus, for humans, it is an essential nutrient. TPP acts in vivo as the coenzyme of enzymes executing several vital metabolic processes such as pentose phosphate pathway and the TCA cycle. Thiamine is on the World Health Organization's List of Essential Medicines that lists the most important medications needed in a basic health system and thiamine deficiency is a widespread health problem. The commercially relevant form of thiamine is un-phosphorylated, since this is the most stable form that can be assimilated and phosphorylated by humans and animals to produce the biologically active cofactor TPP.
The thiamine biosynthetic pathways characterized in bacteria, some protozoans, plants, and fungi, share some common features (FIG. 1). The thiazole and pyrimidine moieties are biosynthesized separately. The pyrimidine moiety, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), is derived from 5-aminoimidazole ribotide (AIR), an intermediate in the de novo purine biosynthetic pathway. In Gram-negative bacteria, conversion of AIR to HMP-P is catalyzed by the thiC gene product. HMP-P is then phosphorylated to HMP-PP by ThiD kinase prior to coupling with the thiazole unit. The thiazole moiety, 5-(2-hydroxyethyl)-4-methylthiazole phosphate (HET-P), is derived from L-tyrosine and 1-deoxy-D-xylulose phosphate (DXP) and cysteine; where the sulfur atom likely derives from L-cysteine. This latter reaction requires expression of at least five genes thiF, thiS, thiG, thiH and thiI.
The pyrimidine and thiazole moieties are then combined to form TMP by the action of thiamine-phosphate synthase (EC 2.5.1.3) encoded by thiE. Thus TMP is the first product of all known thiamine biosynthetic pathways. In E. coli and other Enterobacteriaceae, TMP may be phosphorylated to the cofactor TPP by a thiamine-phosphate kinase (EC 2.7.4.16) encoded by thiL in the presence of ATP. Some bacteria and eukaryotes, rely on the salvage pathway from HMP and HET which requires ThiD, ThiE and ThiM. E. coli uses a salvage enzyme, thiamine kinase, encoded by thiK to convert exogenous thiamine, taken up by the cell, into TMP.
In view of their capacity to synthesize TMP and TPP, micro-organisms can be used as cell factories for the recombinant production of this vitamin. Recombinant production of essential medicines such as the vitamin thiamine requires the use of a suitable host that is capable of producing and exporting thiamine. Un-phosphorylated thiamine (THI) is the desired target for biological production, not only for commercial reasons, but also because thiamine easily crosses cell membranes, in contrast to its phosphorylated forms, and therefore accumulates outside of the production host during fermentation, where it is the most stable form.
The advantages of recombinant Escherichia coli as a cell factory for production of bioproducts are widely recognized due to the fact that: (i) it has unparalleled fast growth kinetics; with a doubling time of about 20 minutes when cultivated in glucose-salts media and under optimal environmental conditions, (ii) it easily achieves a high cell density; where the theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight/1 or roughly 1×1013 viable bacteria/ml. Additionally, there are many molecular tools and protocols at hand for the high-level production of bioproducts in E. coli; heterologous proteins can easily be expressed in E. coli and there are many specialty strains available for the production of specialist end-products.
In most bacteria, TPP is produced by the direct conversion of TMP to TPP, where cell growth requires an intracellular or extracellular supply of TPP. Thus the use of E. coli as a cell factory for thiamine production requires a genetically modified strain that is both viable and at the same time is capable of producing and releasing thiamine into the extracellular environment.